1. Field of the Invention
The present invention relates to biotinylated oligonucleotides and reagents for preparing biotinylated oligonucleotides.
2. Discussion of the Background
The use of labeled oligonucleotides as hybrization probes is widespread. Although labeling of the probes with the isotope .sup.32 P is common, such probes may also be labeled with biotin, which may be detected as a result of its binding to avidin and streptavidin. The use of a biotin-labeled (biotinylated) probe avoids the problems encountered with radioactive labels such as .sup.32 P.
Recently there have been reports of oligonucleotides to which biotin has been covalently attached to the 5'-hydroxyl group (see: Chollet et al, Nucleic Acids Res., vol. 13, p. 1529 (1985); Wachter et al. Nucleic Acids Res., vol. 14, p. 7985 (1986); Agarawal et al, Nucleic Acids Res., vol. 14, p. 6227 (1986); Urdea et al, Nucleic Acids Res., vol. 16, p. 4937 (1988); and Cook et al, Nucleic Acids Res., vol. 16, p. 4077 (1988). However, all of these methods required special reagents and considerable time and effort.
Biotinylated oligonucleotides have also been demonstrated to have utility in ligase-medicated gene detection (Landergreu et al, Science. vol. 241, p. 1077 (1988)), sequencing after amplification by the polymerase chain reaction (PCR) (Mitchell et al, Anal. Biochem., vol. 178, p. 239 (1989)), and nonradioactive sequencing of DNA (Richterich, Nucleic Acids Res., vol. 17, p. 2181 (1989)).
Cocuzza has reported reagents, which can be used in conjunction with automated solid state synthetic technique, for the preparation of 5'-biotinylated oligonucleotides (Cocuzza, Tetrahedron Letters, vol. 30, pp. 6287-6290 (1989) and U.S. Pat. No. 4,908,453). However, the biotinylated oligonucleotides afforded by this approach are not stable to the standard workup procedures required to remove the protecting groups commonly used in solid state oligonucleotide synthesis.
Alves et al have also described a chemical method of labelling oligonucleotides with biotin (Alves et al, Tetrahedron Letters, vol. 30, pp. 3089-3092 (1989)). However, the exemplified reagents possess a short linkage between the phosphoramiditic group and the biotinyl residue.
Furthermore, both the reagents above utilize solvents not ordinarily employed for DNA synthesis, namely dimethylformamide (Cocuzza) and a mixture of dichloroethane and acetonitrile (Alves et al), a further disadvantage in both reagents.
Miyoshi et al (U.S. Pat. No. 4,605,735) disclose a process for preparing oligonucleotide derivatives in which biotin is bonded to the terminal amino group of an oligonucleotide derivative. However, the method of coupling biotin to the DNA requires a separate manual synthesis reaction, after prior preparation of the DNA oligonucleotide.
Carr et al (European Patent Application 202,758) disclose a solid-state method for preparing 5'-biotinylated oligonucleotides by reacting the deprotected 5'-hydroxy group with a phosphorylated biotin derivative. Kempe et al (Nucleic Acids Res., vol. 13, p. 45 (1985)) also disclose a solid state synthesis of 5'-biotinylated oligonucleotides, in which the deprotected 5'-hydroxy group is reacted with p-chlorophenylphosphoditriazolide, the product is hydrolyzed, and the hydrolyzed product is reacted with 2-(biotinylamido)ethanol. Both of these methods suffer from the disadvantages of requiring special reagents. Further, in the case of Kempe et al, the product contains the labile amide linkage.
Levenson et al (U.S. Pat. No. 4,751,313) disclose a method for labelling an oligonucleotide with biotin using a biotin derivative which contains an alkylating intercalation moiety. However, the product of this process is not a 5'-biotinylated oligonucleotide
Ward et al (U.S. Pat. No. 4,711,955) disclose the preparation of labeled nucleotides in which biotin is bonded to the nucleotide via a base (purine, 7-deazapurine, or pyrimidine moiety). Again, the products are not 5'-biotinylated oligonucleotides.
Accordingly, there remains a need for 5'-biotinylated oligonucleotides and reagents for their preparation which do not suffer from the above-mentioned drawbacks.